One of the most successful intracellular bacteria is Wolbachia, harbored by thousaneds of arthropods and nematodes. Wolbachia have recently been shown cause blindness in humans upon release from their nematode host Onchocerca volvulus. Wolbachia have also been shown to infect the well-established model organism Drosophila melanogaster. Wolbachia rely upon maternal transmittance to spread throughout nematode and arthropod populations. To ensure maternal inheritance, Wolbachia must become incorporated into the reproductive germline cells. This study will be the first to define the molecular basis of Wolbachia inheritance in Drosophila. Three specific aims will be pursued: Aim 1 will define the mechanism used by Wolbachia to promote maternal inheritance. Preliminary work indicates that Wolbachia co-localize at the oocyte posterior with germline determinants that give rise to germline cell formation in the embryo. We hypothesize that Wolbachia co-opt the two-step process used for localization of germline determinant proteins: microtubule- based transport and actin-based anchoring at the posterior cortex. To define the mechanism of Wolbachia posterior localization, we will test Wolbachia distribution in mutant backgrounds that disrupt germline determinant localization. Aim 2 will probe molecular interactions between Wolbachia and host. We hypothesize that Wolbachia modify the host protein composition of the vacuole membrane surrounding them to promote maternal inheritance of Wolbachia. To identify Wolbachia-induced modifications to the vacuole membrane, we will perform gradient sedimentation of organelles followed by mass spectrometry. Aim 3 will test the impact of Wolbachia-induced vacuole modifications on Wolbachia inheritance. Candidate host genes likely to elucidate linkage between Wolbachia and posterior determinants or the host cytoskeleton will be the top priority. Wolbachia localization, motility and quantity will be examined in oocytes mutant for those candidate genes. A subset of functionally host relevant proteins will be tested with deletion constructs and GFP-fusions to identify the domains important for Wolbachia localization as well as when/where the host proteins are recruited to Wolbachia-containing vacuoles. This is an exciting time to enter the Wolbachia research. With the direct relevance to human disease and the potential to explore host-microbe interactions from the perspective of Drosophila, studies of Wolbachia are of increasing interest to public health. The long-term goal of this project is to contribute to the community-wide objective of finding new, more effective strategies for curing bacterial diseases.